Protein Sample Preparation - Western Bloting (Jan/25/2010 )

Hi,
I'm facing problem in my protein sample preparation from long time.
Method I use-
1. Harvesting adrenal , pancreas and testis from mouse.
2. Homogenizing with Lysis buffer (containing lysis buffer + triton 0.1 X + Protease Inhibiter )
adding 4 ml 0f Lysis buffer per 1 gram of tissue.
3. Incubation @ 4 degree from 1 hour
4. centrifuge for 30 min on 25000 rpm @ 4 degree
5. After collection supernatant , I add sample loading buffer 5 micro L per 20 micro L of sample.
6. 5 Min. heating in water bath.

Result : No bands in western or dirty picture.

Thanks

Hi there


Are you sure the WB technique is working? (you have other gels working)

Did you check that you had proteins in your lisate? (with bradford or something like that)
Did you check that your transfer was ok? (staining the gel)
Did you check any other protein in your blot? (tubulin)

Which protein are you trying to detect?